HNF3-mediated chromatin remodeling: A role in liver gene regulation

HNF3 介导的染色质重塑：在肝脏基因调控中的作用

• 批准号: 7572861
• 负责人: Sarah E Kohler
• 金额: $ 5.01万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7572861
• 关键词: Address; Affect; Albumins; Binding; Binding Sites; Biological Assay; Cell Differentiation process; Cell Line; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; DNA; DNA Binding Domain; Data; Defect; Deoxyribonuclease I; Deoxyribonucleases; Development; Embryonic Development; Endoderm; Enhancers; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Hepatocyte; Histones; In Vitro; Lead; Liver; Malignant Neoplasms; Mediating; Metabolism; Micrococcal Nuclease; Modeling; Mutate; Mutation; Nucleosomes; Organ; Organogenesis; Physiological; Play; Prealbumin; Primitive foregut structure; Process; Promoter Regions; Regulation; Research; Role; Series; Structure; Teratocarcinoma; Testing; Tretinoin; Up-Regulation; Visceral; alpha-Fetoproteins; base; cell type; chromatin immunoprecipitation; chromatin remodeling; hepatocyte nuclear factor; in vivo; interest; knock-down; precursor cell; research study; small hairpin RNA; transcription factor

DESCRIPTION (provided by applicant): During early development, differentiating cells must activate genes previously not expressed that are required for cell type specification and organogenesis. For these genes to be turned on, chromatin must be de-compacted to allow transcription factors access to the DNA template. The goal of this research is to study the underlying mechanisms involved in chromatin remodeling. Specifically, we will examine chromatin opening at the albumin enhancer by the liver enriched transcription factor, HNF3, in a model of liver development. HNF3 is a pioneer transcription factor that can bind to and open compacted chromatin at the albumin enhancer in vitro in the absence of chromatin remodeling complexes. In order to establish the physiological relevance of this phenomenon, we plan to examine the role of HNF3 in chromatin remodeling in vivo using the F9 teratocarcinoma cell line. These cells, in the presence of retinoic acid, differentiate into visceral endoderm and turn on albumin gene expression as they differentiate. Because we are interested in examining the activation of the albumin enhancer, these cells will serve as a good model to ask questions about the role of HNF3 in chromatin opening. In order to determine changes in chromatin opening, we will use DNase I, micrococcal nuclease (MNase) and chromatin immunoprecipitation (ChIP) assays and compare chromatin accessibility to albumin gene upregulation. By mutating HNF3 binding sites on the albumin enhancer, we can determine whether HNF3 binding is required for chromatin remodeling at the albumin enhancer in vivo. Additional experiments with short hairpin RNAs (shRNAs) against HNF3 will determine whether HNF3 transcriptional activity is required to open chromatin at the albumin enhancer. In addition to determining the role of HNF3 in albumin enhancer remodeling, we will also examine a potential role for HNF3 in the activation of other liver specific genes, including transthyretin and alpha-fetoprotein. By fully understanding the mechanisms involved in gene activation during normal development, we can hopefully better understand aberrations in this process that may lead to developmental defects and cancer.

描述（由申请人提供）：在早期发育过程中，分化细胞必须激活先前未表达的基因，这些基因是细胞类型规范和器官发生所需的。为了打开这些基因，染色质必须被解压缩，以便转录因子能够接触到 DNA 模板。这项研究的目的是研究染色质重塑的潜在机制。具体来说，我们将在肝脏发育模型中检查肝脏富集转录因子 HNF3 在白蛋白增强子处的染色质打开情况。 HNF3 是一种先驱转录因子，在没有染色质重塑复合物的情况下，可以在体外与白蛋白增强子处结合并打开压缩的染色质。为了确定这种现象的生理相关性，我们计划使用 F9 畸胎癌细胞系检查 HNF3 在体内染色质重塑中的作用。这些细胞在视黄酸存在下分化为内脏内胚层，并在分化时开启白蛋白基因表达。因为我们对检查白蛋白增强子的激活感兴趣，所以这些细胞将作为一个很好的模型来询问有关 HNF3 在染色质开放中的作用的问题。为了确定染色质开放的变化，我们将使用 DNase I、微球菌核酸酶 (MNase) 和染色质免疫沉淀 (ChIP) 检测，并比较染色质可及性与白蛋白基因上调的关系。通过突变白蛋白增强子上的 HNF3 结合位点，我们可以确定体内白蛋白增强子处的染色质重塑是否需要 HNF3 结合。针对 HNF3 的短发夹 RNA (shRNA) 的其他实验将确定是否需要 HNF3 转录活性来打开白蛋白增强子处的染色质。除了确定 HNF3 在白蛋白增强子重塑中的作用外，我们还将研究 HNF3 在其他肝脏特异性基因（包括转甲状腺素蛋白和甲胎蛋白）激活中的潜在作用。通过充分了解正常发育过程中基因激活的机制，我们有望更好地理解这一过程中可能导致发育缺陷和癌症的畸变。